Oils with anti-inflammatory activity containing natural specialized proresolving mediators and their precursors

ABSTRACT

The present invention encompasses oils that have anti-inflammatory or resolution-stimulating activity that contain or are enriched with Specialized Proresolving Mediators (SPM) or SPM precursors, which originate from an oil obtained from organisms containing long chain omega-3 polyunsaturated fatty acids, such as fish, crustaceae, algae, and mollusks. The invention also encompasses a method for the production of these oils, and the utilization of the oils for nutritional supplements, pharmaceutical formulations, and cosmetic formulations, which can be employed for treating an inflammatory condition.

This application is a continuation of U.S. patent application Ser. No.16/882,910, filed May 26, 2020, which is a continuation of U.S. patentapplication Ser. No. 16/297,240 filed on Mar. 8, 2019, which is acontinuation of U.S. patent application Ser. No. 15/935,913 filed onMar. 26, 2018, which is a continuation of Ser. No. 14/400,198, filed onNov. 10, 2014 (now U.S. Pat. No. 10,568,858), which is a 35 USC § 371U.S. National Stage Application of International Patent Application No.PCT/US2013/040314, filed on May 9, 2013, entitled, “OILS WITHANTI-INFLAMMATORY ACTIVITY CONTAINING NATURAL SPECIALIZED PRORESOLVINGMEDIATORS AND THEIR PRECURSORS,” which claims priority to U.S.Provisional Patent Application No. 61/645,281, filed on May 10, 2012,the entire contents of each of which are incorporated herein byreference and relied upon.

FIELD OF THE INVENTION

The invention relates generally to the fields of natural products,inflammation, pathology, and medicine. More particularly, the inventionrelates to Specialized Proresolving Mediators (SPMs) and SPM precursorsobtained from natural sources, and their use in nutritional supplementsand pharmaceutical and cosmetic formulations for amelioratinginflammation and diseases having an inflammatory component.

BACKGROUND OF THE INVENTION

Inflammation is a complex biological response that animals make inattempt to remove or neutralize pathogens, irritants, or cell damage,and to initiate healing of injured tissues. The classical physicalsymptoms of inflammation include dolor (pain), calor (heat), rubor(redness), tumor (swelling), and functio laesa (loss of function).Initiation of an inflammatory response is associated with the activationof polymorphonuclear leukocytes (neutrophils), monocytes, and tissuemacrophages. Activation of these cells unleashes a cascade ofpro-inflammatory signaling events mediated by various small moleculesand peptides, including prostaglandins, leukotrienes, chemokines andcytokines, and activated complement factors. These signaling eventsstimulate cellular chemotaxis, endothelial permeability, vasodilation,stimulation of sensory nerves, and activation of coagulation, which inturn lead to the physical symptoms of inflammation. Of importance, it isnow understood that also the termination of inflammation, resolution, isan actively regulated part of the inflammatory response which involves acoordinated set of cellular and molecular events in order to restoretissue structure and function.

While inflammation is beneficial and indeed required for good health, itcan also go awry and cause disease. For example, reperfusion injuryfollowing ischemia (e.g., in myocardial infarction or ischemic stroke)stimulates an acute inflammatory response that can damage tissue. Andwhen a normal inflammatory response fails to terminate (resolve) alterremoval of the original stimulus, chronic inflammation can ensue.Chronic inflammation damages healthy tissue and can cause or aggravate anumber of different diseases including, e.g., atherosclerosis and otherdiseases of the vascular system, asthma, acne, psoriasis, rheumatoidarthritis, chronic obstructive pulmonary disease, cystic fibrosis,inflammatory bowel disease, and different kinds of autoimmune disease.Chronic inflammation has also been associated with type-2 diabetes,obesity, Alzheimer's disease, and cancer.

The resolution of inflammation is now recognized to constitute an activephysiological process that forms an integral part of the inflammatoryresponse. Resolution as the disappearance of the inflammatory exudate,and restoration of proper tissue structure and function, is mediated byseveral different molecular and cellular mechanisms. These include theclearance and metabolic destruction of inflammatory cytokines; formationof anti-inflammatory mediators such as transforming growth factor-beta,interleukin-10, annexin Al, and lipoxin A4; apoptosis ofpro-inflammatory neutrophils; active recruitment of immunoregulatorymonocytes/macrophages and eosinophils; and efferocytosis and egress ofinflammatory leukocytes. Of particular relevance, it has been discoveredthat a family of substances collectively named Specialized ProresolvingMediators (SPMs) are central regulators of resolution. SPMs have potentanti-inflammatory activities (namely they reduce neutrophilinfiltration), actively stimulate the removal and disappearance of theinflammatory exudate, expedite clearance of infection, and stimulatewound healing. SPMs are a genus of recently characterized lipidmediators identified in resolving exudates of inflammatory lesions, andcomprise enzymatically oxygenated derivatives of long chainpolyunsaturated fatty acids such the omega-3 polyunsaturated fatty acids(ω-3 PUFA) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA).SPMs have potent agonistic activities on specific G protein-coupledreceptors, thereby activating different aspects of the resolution ofinflammation. The SPMs consist of several different families of longchain ω-3 PUFA-derived lipid mediators: resolvins, protectins andmaresins—members of each of which control the duration and magnitude ofinflammation by stimulating endogenous resolution mechanisms (Bannenberg& Serhan, 2010).

The biosynthesis of SPMs involves the positional and stereospecificincorporation of one or two molecules of molecular oxygen into apolyunsaturated fatty acid, catalyzed by substrate- andpositionally-selective fatty acid oxygenases such as lipoxygenases,cyclooxygenase type-2 when acetylated by aspirin, and several cytochromeP450 oxidases. The PUFA which are currently best understood to act assubstrate for the formation of SPMs are EPA and DHA.

The first step in the endogenous formation of SPMs involves theenzymatic oxygenation of a long chain ω-3 PUFA in astereochemically-defined manner leading to the formation of specificfatty acid hydroperoxides. The fatty acid hydroperoxides can betransformed to SPMs via several biosynthetic routes. The first is areduction of the hydroperoxyl group to form the correspondingmonohydroxylated fatty acid. Some of these monohydroxylated productsfunction as intermediate precursors for a subsequent enzymaticoxygenation to form dihydroxylated and trihydroxylated SPMs. Forexample, 17-hydroxy-docosahexaenoic acid (17-HDHA), the product of a15-lipoxygenase-catalyzed oxygenation with DHA, is the substrate for theformation of four distinct trihydroxylated resolvins RvD1, RvD2, RvD3,and RvD4. In this manner, 17-HDHA can be considered an SPM precursor. Ina different biosynthetic pathway, the first formed fatty acidhydroperoxide can rearrange enzymatically to form an epoxide and isthereafter hydrolyzed enzymatically, to form a dihydroxylated product.Examples of such dihydroxylated lipid mediators are protectin D1 andmaresin 1.

EPA and DHA thus constitute endogenous substrates in the bodies ofanimals and humans from which the in vivo formation proceeds to formEPA- and DHA-derived resolvins (so called E-series and D-seriesresolvins, respectively), and DHA-derived protectins and maresins, whichare dihydroxylated and trihydroxylated EPA and DHA derivatives withpotent anti-inflammatory and resolution-activating activity in vivo(Bannenberg and Serhan, 2010). The resolvins, protectins, and maresinsare SPMs and act as endogenous receptor ligands or allosteric modulatorsto potently activate cellular responses that concertedly activateanti-inflammatory actions and expedite, stimulate, and trigger theresolution of inflammation. Furthermore, several enzymatically formedepoxide derivatives of EPA and DHA are now also known to possess potentanti-inflammatory activity themselves as well (Wagner, 2011) and areconsidered as SPMs here. Prior literature has also described thepresence of several PUFA-derived lipid mediators in their freecarboxylic acid form in cells and tissue of trout and anchovy (Pettitt,1989; Hong, 2005; Oh, 2011; Raaz, 2011). The formation of SPMs occursendogenously within the bodies of organisms, in several tissues and celltypes, and occurs intracellularly. The substrate for SPM formation arethe free carboxylic acid forms of EPA and DHA; these free fatty acidshave been liberated by a phospholipase from membrane phospholipidscontaining EPA and DHA. There is no prior description that SPMs whichare naturally formed within the cells or tissues of living organisms canbe found outside the body of an animal or human being.

Several SPMs have now been synthesized by chemical synthesis methods.The synthetic SPMs have been instrumental in the delineation of thechemical structures and activity of the SPMs formed by cells in thebodies of animals. Also, structural analogues of SPMs have beensynthesized by chemical synthesis methods. The advantage of syntheticforms of SPMs is their well-controlled purity. However, chemicalsynthesis of SPMs is a technically challenging and expensive process,since it is difficult to obtain the precise stereochemistry and doublebond geometries that are important for bioactivity. It is therefore ofhigh interest to have access to and obtain large quantities of thenaturally bioactive forms of the SPMs.

Of particular relevance to the current invention, some of themonohydroxylated and epoxygenated derivatives constitute biosyntheticintermediates with more potent anti-inflammatory activity than EPA andDHA since they are more proximate intermediates in the biosynthesis ofseveral SPMs than EPA and DHA themselves. These intermediate precursorsare therefore considered SPM precursors.

It is of interest to note that several other long chain omega-3 PUFA,such as docosapentaenoic acid (ω-3), can also be transformed intooxygenated derivatives by the same oxygenases, with some derivativeshaving marked anti-inflammatory activity. There are also long chainomega-6 PUFA-derived anti-inflammatory and resolution-stimulating(proresolving) lipid mediators, such as lipoxin A4 formed through twoenzymatic oxygenation steps from arachidonic acid, prostaglandin D2formed from arachidonic acid by cyclooxygenases which gives rise todehydration products with potent anti-inflammatory activity, and lipidmediators derived from docosapentaenoic acid (ω-6) withanti-inflammatory activities. In this respect it is important tounderstand that also arachidonic acid is an essential long chain PUFA,like EPA and DHA, and is usually present in all organisms that alsocontain long chain ω-3 fatty acids.

Even though the chemical structures of several SPMs are now known andtheir anti-inflammatory and pro-resolving activities have been studiedin some detail in different experimental models of inflammation, nonutritional supplement, cosmetical formulation, or approvedpharmaceutical formulation that contains an SPM has been developed forthe inhibition or resolution of inflammation.

Because increased blood levels of EPA and DHA are associated withdecreased incidence of, and propensity to develop, cardiovasculardisease, the oral supplementation of omega-3 PUFA-containing oils isincreasingly being used to ameliorate inflammation with some degree ofsuccess. The anti-inflammatory potential of dietary long chain ω-3 PUFAis widely believed to be related to the increase in tissue levels of EPAand DHA. Augmenting endogenous levels of EPA and DHA is commonlybelieved to favor an anti-inflammatory status through competition forthe endogenous formation of the inflammation-activating eicosanoidsderived from the omega-6 PUFA arachidonic acid (AA), the formation ofEPA- and DHA-derived 3-series prostaglandins and thromboxane with muchlower inflammatory potency and efficacy, and biophysical changes withinmembrane domains and membrane proteins which modulate immune cellfunction. However, recent research has shown that long chain (ω-3 PUFAare serving as endogenous substrates for the enzymatic formation ofendogenous SPMs which act as autacoids to functionally antagonizeinflammation and which actively expedite resolution. This recentrecognition that EPA and DHA act as the physiological substrate for theformation of autacoids which drive the resolution of inflammation,affords renewed understanding of the essential nature of long chain ω-3PUFA for human health. It is now well established that increasedconsumption of EPA and DHA-containing foods increase the tissue levelsof these 0-3 PUFA. More recently, it has been shown that dietarysupplementation with EPA and DHA indeed permits a measurable increase inthe endogenous formation of some EPA- and DHA-derived oxygenated lipidmediators in humans (Anta, 2005; Shearer, 2010; Mas, 2012).

Long chain polyunsaturated fatty acids containing an omega-3 double bondare naturally formed by algae and other microorganisms forming the basisof the biotrophic chain of transfer of long chain ω-3 fatty acids suchas EPA and DHA (Gladyshev, 2013) Mammals depend on the adequate supplyof EPA and especially DHA through dietary sources, mainly throughconsumption of fish containing significant tissue levels of EPA and DHAwhich have upon their turn obtained these essential PUFA from the foodchain. Mammals including man can endogenously synthesize EPA and DHAfrom alpha-linolenic acid, however the efficiency of this conversion isvery limited and not adequate for the requirements for EPA and DHA.Dietary intake of EPA- and DHA-containing foods, and dietarysupplementation with oils containing significant levels of EPA and DHA,are currently viewed as appropriate means to obtain a daily intake thatcan significantly increase the levels of long chain ω-3 PUFA and therebyattain an increased capacity to lower the intensity and duration ofinflammatory reactions and disease.

Dietary requirements vary with age and life stage, and the essentialnature of EPA and DHA for human health is therefore conditional.Circumventing the dependence of the substantial human need for longchain ω-3 PUFA on the natural food chain and growing global humandemands for long chain ω-3 PUFA sufficiency, recent progress inbiotechnology has permitted the creation of e.g. transgenic plants andmicroorganisms endowed with the biosynthetic capacity to form long chainω-3 PUFA such as EPA and DHA (Perie, 2012).

Dietary supplementation with oils containing long chain ω-3 PUFA iscurrently achieved by consumption of formulations which encompass manydifferent presentations. The oils currently employed consist for thelargest part (in volumes consumed) of EPA- and DHA-containing oilsextracted from fish, of which the Peruvian anchovy makes up asubstantial part. Other oils include those extracted from e.g. salmonand tuna. There is available a variety of different grades of oilsranging from oils which have been cold-pressed and which have undergonevery few steps to only clear the oil from color or odorous substancespresent in the oil, to oils which have been selectively concentratedtowards obtaining a specific long chain ω-3 fatty acid. Fish oilscontaining modest concentrations of long chain ω-3 PUFA (usually up toapproximately 30%), or with concentrations increased by distillation toapproximately 55%, are used widely in nutritional supplements for thetreatment of, for example, hypertriglyceridemia, and for vascular andeye health. One good example of a long chain ω-3 PUFA concentrate madefrom fish oil which can currently be produced at industrial scale forthe pharmaceutical sector contains 97% EPA in the form of an ethylester.

General methods involving lipid chemistry, industrial processes relatingto oils and fatty acids, and conventional pharmaceutical sciences, aredescribed in: (Remington, 2005; Martinez, 2007; Gunstone & Padley, 1997;Shahidi, 2005).

The fish oil industry currently manufactures a range of different EPA-and DHA-containing oil grades. EPA and DHA-containing oils are alsoextracted from other organisms such as krill, squid, algae, yeasts,protozoa, and from transgenic plants endowed with genes coding forenzymes that permit the biosynthesis of EPA and DHA and other long chainω-3 PUFA such as stearidonic acid (SDA). Formulations available on themarket for human consumption range from oils as such, encapsulated oils,emulsions, and stabilized powders. In all cases, the objective is toprovide dietary supplements and pharmaceutical ingredients which aim toprovide sufficiently high doses to humans to aid in augmentingendogenous tissue levels of EPA and DHA. Although relatively rapidabsorption and redistribution of EPA and DHA into specific cell types,platelets and lipoproteins in the circulation can be measured (within 24hours), it is generally accepted that the health-promoting actions ofEPA and DHA upon oral consumption need significant time due to thesupposed requirement that increased tissue levels of EPA and DHA need tobe build up and which takes several weeks to months of taking doses ofat least several hundreds of milligrams of EPA and DHA every day.

A characteristic of the need to provide EPA and DHA as essentialnutrients for lowering inflammatory reactions, and preventing andtreating inflammatory conditions, is that the endogenous enzymaticconversion of EPA and DHA, attained by dietary food intake and specificsupplementation, to SPMs is a multistep enzymatic process which involvesthe liberation of phospholipid-bound EPA and DHA by phospholipases,followed by one or more enzymatic oxygenation reactions catalyzed byspecific fatty acid oxygenases to form the active SPMs. These processesfunction adequately under healthy conditions, however low EPA and DHAtissue levels, as well as limited or inadequate conversion in thetissues of the body of long chain polyunsaturated fatty acids to SPMsare considered to contribute to, predispose to, or underlie inflammatoryconditions and exaggerated inflammatory reactions.

BRIEF DESCRIPTION OF THE INVENTION

The invention is based on the surprising discovery that SpecializedProresolving Mediators (SPMs) and SPM precursors are present in oilsextracted from organisms containing long chain ω-3 PUFA. Oils containingat least one SPM or SPM precursor and having anti-inflammatory orresolution-stimulating (proresolving) activity can be produced using amethod comprising the steps of measuring the presence or level of SPMsor SPM precursors in a long chain ω-3 PUFA-containing oil (such as acrude, refined, or concentrated long chain ω-3 fatty acid-containingoil), fractionating the oil into a plurality of fractions, measuring theanti-inflammatory or resolution-stimulating activity of the oilfractions, and optionally repeating these three steps, to obtain an oilthat contains or is enriched with at least one SPM or SPM precursor andhas anti-inflammatory or resolution-stimulating activity. The SPMs andSPM precursors can be found in the form of saponifiable substances. Theoils can furthermore contain long chain ω-3 PUFA, such as EPA and DHA.

Technologies which can be employed for such fractionation includeextraction and separation methods. Technologies that are of particularinterest for obtaining oils containing or enriched with SPMs and SPMprecursors are supercritical fluid extraction (SFE) and supercriticalfluid chromatography (SFC) employing carbon dioxide as solvent. Theadministration to subjects of an effective amount of these oilsconstitutes a method of reducing inflammation or stimulating theresolution of inflammation. The oils can be used for the manufacturingof nutritional supplements, pharmaceutical formulations, and cosmeticalformulations, comprising an effective amount of an oil withanti-inflammatory or resolution-stimulating activity. These supplementsand formulations thus constitute anti-inflammatory and proresolvingcompositions that can be manufactured in large quantities and do notrequire the addition of expensive chemically-synthesized SPMs.

Although methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present invention,suitable methods and materials are described below. All patents, patentapplications, and publications mentioned herein are incorporated byreference in their entirety. In the case of conflict, the presentspecification, including definitions will control. In addition, theparticular embodiments discussed below are illustrative only and notintended to be limiting. Other aspects of the present invention will beevident for a person skilled in the art in view of the description ofthe invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects, features and advantages of the presentinvention will be more clearly understood from the following detaileddescription taken in conjunction with the accompanying drawings, inwhich:

FIGS. 1A-1H shows the effect alter oral administration of a series ofconsecutively eluted oil fractions (number 1-8, respectively), obtainedby industrial-scale SFC fractionation of an intermediate long chain ω-3PUFA-ethyl ester concentrate (containing 70% EPA-ethyl ester (EE) andDHA-EE combined), on acute inflammatory changes occurring subcutaneouslyin mice induced by subcutaneous (s.c.) administration oflipopolysaccharide (LPS).

FIG. 2 shows the relative abundance of the ethyl-esterified andsaponifiable forms of various monohydroxylated derivatives of thepolyunsaturated fatty acids EPA and DHA, in consecutively-eluted oilfractions of an industrial scale SFC fractionation of an intermediatelong chain ω-3 PUFA-ethyl ester concentrate (containing 70% EPA-EE andDHA-EE combined). The fractions numbered 1-8 are the same as thosetested for anti-inflammatory activity as shown in FIG. 1.

FIG. 3A. shows the concentration of the ethyl ester of the D-seriesresolvin precursor 17-HDHA in several consecutively eluted oil fractionsof industrial scale SFC of an intermediate long chain ω-3 PUFA-ethylester concentrate (containing 70% EPA-EE and DHA-EE combined),corresponding to the same fractions as shown in FIGS. 1 and 2.

FIG. 3B. shows the results of a chiral high performance liquidchromatography-triple quadrupole mass spectrometric analysis of the17-HDHA-ethyl ester found enriched in fraction 1, carried out in orderto determine the relative abundance of the stereoisomers 17S-HIDHA and17R-HDHA in the ethyl ester oil fractions 1-8, obtained alter alkalinehydrolysis (top panel).

FIG. 4 shows the anti-inflammatory effect of oil fraction 1 and 17S-HDHAadministered by gavage in a murine model of peritoneal inflammationinduced by intraperitoneal administration of the yeast membrane extractzymosan A.

FIGS. 5A-5C show the presence of several specific SPMs and SPMprecursors in different oil fractions.

FIGS. 6A-6B, show that selective enrichment of specific SPMs and SPMprecursors can be achieved by SFC fractionation of a long chain cω-3PUFA concentrate.

FIG. 7 shows the resolution of inflammation stimulated by a SPMprecursor-containing oil.

DETAILED DESCRIPTION OF THE INVENTION

It has been discovered that SPMs and SPM precursors are contained inoils derived from natural sources including fish, crustaceae (krill),algae (long chain ω-3 PUFA-producing algae), mollusks, and from otherorganisms containing long chain ω-3 PUFA. This permits the production ofoils with anti-inflammatory and resolution-stimulating activitypurposefully containing or enriched with one or more SpecializedProresolving Mediators (SPMs) and SPM precursors from natural sources,as well as nutritional supplements, pharmaceutical formulations, andcosmetical formulations containing these oils, and methods of using suchsupplements and formulations to treat or prevent inflammatory conditionsand diseases associated with inflammation, by inhibiting inflammation orstimulating the resolution of inflammation. The below describedembodiments illustrate representative examples of these methods andcompositions. Nonetheless, from the description of these embodiments,other aspects of the invention can be made and/or practiced based on thedescription provided below. Unless otherwise defined, all technicalterms used herein have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs.

A first aspect of the present invention is formed by oils that haveanti-inflammatory or resolution-stimulating activity characterized inthat they contain or are enriched with at least one SPM or SPMprecursor, wherein the SPM or SPM precursor originates from an oilobtained from organisms containing long chain ω-3 fatty acids.

As used in the present invention, the term “enriched” refers to an oilcontaining Specialized Proresolving Mediators (SPMs) and/or SPMprecursors when it contains a higher level of SPMs and/or SPM precursorsthan the source from which it was made.

As used in the present invention, the term “Specialized ProresolvingMediator (SPM)” relates to a PUFA-derived enzymatically-oxygenatedderivative which has potent anti-inflammatory and resolution-activatingactivity and which acts as endogenous regulator of the inflammatoryresponse to bring an inflamed tissue back towards its non-inflamed andhealthy state. SPMs act as endogenous receptor ligands or allostericmodulators to potently activate cellular responses that concertedlyactivate anti-inflammatory actions and expedite, stimulate, and triggerresolution of inflammation.

As used in the present invention, the term “SPM precursor” refers to anenzymatically oxygenated derivative of a PUFA which requires anadditional enzymatic reaction to convert it to a SPM. An SPM precursoris a more proximate substrate for the endogenous formation of an SPMthan the corresponding PUFA substrate itself.

These oils contain or are enriched with at least one SPM or SPMprecursor that originates from an oil extracted from organismscontaining long chain (ω-3 PUFA, preferably fish, crustaceae, algae, andmollusks, or other long chain (ω-3 PUFA-containing organisms, such asother marine organisms, plants, microbial organisms, and transgenicorganisms endowed with the capacity to form long chain ω-3polyunsaturated fatty acids.

SPMs that may be present in oils extracted from natural sources includethe following:

-   resolvin E1 (RvE1;    5S,12R,18R-trihydroxy-eicosa-6Z,8E,10E,14Z,16E-pentaenoic acid),-   18S-resolvin E1 (18S-RvE;    5S,12R,18S-trihydroxy-eicosa-6Z,8E,10E,14Z,16E-pentaenoic acid),-   20-hydroxy-RvE 1    (5S,12R,18R,20-tetrahydroxy-eicosa-6Z,8E,10E,14Z,16E-pentaenoic    acid),-   resolvin E2 (RvE2;    5S,18-dihydroxy-eicosa-6E,8Z,11Z,14Z,16E-pentaenoic acid),-   resolvin E3 (RvE3;    17,18R-dihydroxy-eicosa-5Z,8Z,11Z,13E,15E-pentaenoic acid),-   18S-resolvin E3 (18S-RvE3;    17,18S-dihydroxy-eicosa-5Z,8Z,11Z,13E,15E-pentaenoic acid),-   17,18-epoxy-eicosa-5Z,8Z,11Z,13E,15E-pentaenoic acid,-   lipxin A₅ (LXA₅;    5S,6R,15S-trihydroxy-eicosa-7E,9E,11Z,13E,17Z-pentaenoic acid),    15-epi-lipxin A₅ (LXA₅;    5S,6R,15R-trihydroxy-eicosa-7E,9E,11Z,13E,17Z-pentaenoic acid),-   maresin 1 (MaR;    7R,14S-dihydroxy-docosa-4Z,8E,10E,12Z,16Z,19Z-hexaenoic acid),-   7S-maresin 1 (7S-MaR1;    7S,14S-dihydroxy-docosa-4Z,8E,10E,12Z,16Z,19Z-hexaenoic acid),-   7S,14S-diHDHA    (7S,14S-dihydroxy-docosa-4Z,8E,10Z,12E,16Z,19Z-hexaenoic acid),-   protectin D1 (PD1;    10R,17S-dihydroxy-docosa-4Z,7Z,11E,13E,15Z,19Z-hexaenoic acid),-   10S,17S-HDHA    (10S,17S-dihydroxy-docosa-4Z,7Z,11E,13Z,15E,19Z-hexaenoic acid),-   14S,21S-diHDHA    (14S,21S-dihydroxy-docosa-4Z,7Z,10Z,12E,16Z,19Z-hexaenoic acid),-   14S,21R-diHDHA    (14S,21R-dihydroxy-docosa-4Z,7Z,10Z,12E,16Z,19Z-hexaenoic acid),-   14R,21S-diHDHA    (14R,21S-dihydroxy-docosa-4Z,7Z,10Z,12E,16Z,19Z-hexaenoic acid),-   14R,21R-diHDHA    (14R,21R-dihydroxy-docosa-4Z,7Z,10Z,12E,16Z,19Z-hexaenoic acid),-   13 S,14 S-epoxy-DHA    (13S,14S-epoxy-docosa-4Z,7Z,9E,11E,16Z,19Z-hexaenoic acid),-   16,17 S-diHDHA    (16,17S-dihydroxy-docosa-4Z,7Z,10Z,12E,14E,19Z-hexaenoic acid),    16,17-epoxy-DHA (16,17-epoxy-docosa-4Z,7Z,10Z,12E,14E,19Z-hexaenoic    acid), resolvin D1 (RvD1; 7S,8R,17    S-trihydroxy-docosa-4Z,9E,11E,13Z,15E,19Z-hexaenoic acid),-   resolvin D2 (RvD2;    7S,16R,17S-trihydroxy-docosa-4Z,8E,10Z,12E,14E,19Z-hexaenoic acid),-   resolvin D3 (RvD3;    4S,11R,17S-trihydroxy-docosa-5Z,7E,9E,13Z,15E,19Z-hexaenoic acid),-   resolvin D4 (RvD4;    4S,5,17S-trihydroxy-docosa-6E,8E,10Z,13Z,15E,19Z-hexaenoic acid),-   resolvin D5 (RvD5;    7S,17S-dihydroxy-docosa-5Z,8E,10Z,13Z,15E,19Z-hexaenoic acid),-   resolvin D6 (RvD6;    4S,17S-dihydroxy-docosa-5E,7Z,10Z,14Z,16E,19Z-hexaenoic acid),-   aspirin-triggered resolvin D1 (AT-RvD1;    7S,8R,17R-trihydroxy-docosa-4Z,9E,11E,13Z,15E,19Z-hexaenoic acid),-   aspirin-triggered resolvin D2 (AT-RvD2; 7S,16R,17R-trihydroxy-docosa    4Z,8E,10Z,12E,14E,19Z-hexaenoic acid),-   aspirin-triggered resolvin D3 (AT-RvD3;    4S,11,17R-trihydroxy-docosa-5Z,7E,9E,13Z,15E,19Z-hexaenoic acid),-   aspirin-triggered resolvin D4 (AT-RvD4;    4S,5,17R-trihydroxy-docosa-6E,8E,10Z,13Z,15E,19Z-hexaenoic acid),-   aspirin-triggered resolvin D5 (AT-RvD5;    7S,17R-dihydroxy-docosa-5Z,8E,10Z,13Z,15E,19Z-hexaenoic acid),-   aspirin-triggered resolvin D6 (AT-RvD6;    4S,17R-dihydroxy-docosa-5E,7Z,10Z,14Z,16E,19Z-hexaenoic acid),-   7S,17S-diHDPA n-3    (7S,17S-dihydroxy-docosa-8E,10Z,13Z,15Z,19Z-pentaenoic acid (ω-3))-   lipoxin A₄ (LXA₄;    5S,6R,15S-trihydroxy-eicosa-7E,9E,11Z,13E-tetraenoic acid),-   15-epi-lipxin A₄ (15-epi-LXA₄;    5S,6R,15R-trihydroxy-eicosa-7E,9E,11Z,13E-tetraenoic acid),-   delta12-prostaglandin J₂ (delta2-PGJ₂;    11-oxo-15S-hydroxy-prosta-5Z,9,12E-trienoic acid)-   15-deoxy-delta12,14-prostaglandin J₂ (15-deoxy-delta12,14-PGJ₂;    11-oxo-prosta-5Z,9,12E,14E-tetraenoic acid)-   11(12)-epoxy-eicosatetraenoic acid (11(12)-EpETE;    11(12)-epoxy-eicosa-5Z,8Z,14Z,17Z-tetraenoic acid)-   17(18)-epoxy-eicosatetraenoic acid (17(18)-EpETE;    17(18-epoxy-eicosa-5Z,8Z,11Z,14Z-tetraenoic acid)-   19(20)-epoxy-docosapentaenoic acid (19(20)-EpDPE;    19(20)-epoxy-docosa-4Z,7Z,10Z,13Z,16Z-pentaenoic acid)-   10S,17S-HDPA n-6    (10S,17S-dihydroxy-docosa-4Z,7Z,11E,13Z,15E-pentaenoic acid),-   7,17-HDPA n-6 (7,17-dihydroxy-docosa-4Z,8E,10Z,13Z,15E-pentaenoic    acid), 7,14-HDPA n-6    (7,14-dihydroxy-docosa-4Z,8E,10Z,12Z,16Z-pentaenoic acid),-   10S,17S-HDPA n-6    (10S,17S-dihydroxy-docosa-7Z,11E,13Z,15E,19Z-pentaenoic acid), and-   7,17-HDPA n-6 (7,17-dihydroxy-docosa-8E,10Z,13Z,15E,19Z-pentaenoic    acid).    Examples of the presence of these compounds in oils and oil    fractions are shown in Examples 1-3.

Precursors of SPMs that may be present or enriched in oils extractedfrom natural sources include the following. Examples of the presence ofthese compounds in oils and oil fractions are shown in Examples 1-3.

5S-HEPE (5 S-hydroxy-eicosa-6E,8Z,11Z,14Z,17Z-pentaenoic acid); 11S-HEPE(11S-hydroxy-eicosa-5Z,8Z,12E,14Z,17Z-pentaenoic acid); 12S-HEPE (12S-hydroxy-eicosa-5Z,8Z,10E,14Z,17Z-pentaenoic acid); 12R-HEPE(12R-hydroxy-eicosa-5Z,8Z,10E,14Z,17Z-pentaenoic acid); 15S-HEPE (15S-hydroxy-eicosa-5Z,8Z,11Z,13E,17Z-pentaenoic acid); 18S-HEPE(18S-hydroxy-eicosa-5Z,8Z,11Z,14Z,16E-pentaenoic acid); 18R-HEPE(18R-hydroxy-eicosa-5Z,8Z,11Z,14Z,16E-pentaenoic acid); 4S-HDHA(4S-hydroxy-docosa-5E,7Z,10Z,13Z,16Z,19Z-hexaenoic acid); 7S-HDHA(7S-hydroxy-docosa-4Z,8E,10Z,13Z,16Z,19Z-hexaenoic acid); 10S-HDHA(10S-hydroxy-docosa-4Z,7Z,11E,13Z,16Z,19Z-hexaenoic acid); 11S-HDHA(11S-hydroxy-docosa-4Z,7Z,9E,13Z,16Z,19Z-hexaenoic acid); 14S-HDHA (14S-hydroxy-docosa-4Z,7Z,10Z,12E,16Z,19Z-hexaenoic acid); 14R-HDHA(14R-hydroxy-docosa-4Z,7Z,10Z,12E,16Z,19Z-hexaenoic acid); 17S-HDHA(17S-hydroxy-docosa-4Z,7Z,10Z,13Z,15E,19Z-hexaenoic acid); 17R-HDHA(17R-hydroxy-docosa-4Z,7Z,10Z,13Z,15E,19Z-hexaenoic acid); 20S-HDHA(20S-hydroxy-docosa-4Z,7Z,10Z,13Z,16Z,19Z-hexaenoic acid); 17S-HDPAn-6(17S-hydroxy-docosa-4Z,7Z,10Z,13Z,15E-pentaenoic acid); 14S-HIDPAn-6(14S-hydroxy-docosa-4Z,7Z,10Z,12E,16Z-pentaenoic acid); 10S-HDPAn-6 (10S-hydroxy-docosa-4Z,7Z,11E,13Z,16Z-pentaenoic acid); 17S-HDPAn-3(17S-hydroxy-docosa-7Z,10Z,13Z,15E,19Z-pentaenoic acid); 14S-HDPAn-3(17S-hydroxy-docosa-7Z,10Z,12E,16Z,19Z-pentaenoic acid); 10S-HDPAn-6(10S-hydroxy-docosa-7Z,11E,13Z,16Z,19Z-pentaenoic acid); 15S-HETE (15S-hydroxy-eicosa-5Z,8Z,11Z,13E-tetraenoic acid); and/or 15R-HETE(15R-hydroxy-eicosa-5Z,8Z,11Z,13E-tetraenoic acid).

In addition to the foregoing, SPMs and SPM precursors derived from anyof the following omega-3 PUFA or omega-6 PUFA may be present or enrichedin oils extracted from natural sources. These fatty acids may give riseto SPM precursors and SPMs through enzymatic oxygenation.

TABLE 1 Fatty acid name Chemical name Hexadecatrienoic acid (HTA) 16:3(n-3) all-cis-7 ,10,13-hexadecatrienoic acid a-Linolenic acid (ALA) 18:3(n-3) all-cis-9,12,15-octadecatrienoic acid Stearidonic acid (SDA) 18:4(n-3) all-cis-6,9,12,15-octadecatetraenoic acid Nonadecatetraenoic acid19:4 (n-3) all-cis-7 ,10,13,16-nonadecatetraenoic acid Eicosatrienoicacid 20:3 (n-3) all-cis-11,14,17-eicosatrienoic acid Eicosatetraenoicacid 20:4 (n-3) all-cis-8,11,14,17-eicosatetraenoic acidEicosapentaenoic acid (EPA) 20:5 (n-3)all-cis-5,8,11,14,17-eicosapentaenoic acid Heneicosapentaenoic acid 21:5(n-3) all-cis-6,9,12,15,18-heneicosapentaenoic acid Docosapentaenoicacid (DPA) 22:5 (n-3) all-cis-7 ,10,13,16,19-docosapentaenoic acidDocosahexaenoic acid (DHA) 22:6 (n-3) all-cis-4,7,10,13,16,19-docosahexaenoic acid Tetracosapentaenoic acid 24:5 (n-3)all-cis-9,12,15,18,21-tetracosapentaenoic acid Tetracosahexaenoic acid24:6 (n-3) all-cis-6,9,12,15,18,21-tetracosahexaenoic acid

In addition to the listed examples of SPMs and SPM precursors, it can beenvisioned that other mono-, di-, and tri-hydroxylated and epoxygenatedderivatives of the above mentioned polyunsaturated fatty acids maypossess anti-inflammatory and proresolving activities and can be foundto be present and enriched in oils obtained from organism which containlong chain ω-3 PUFA including fish, crustaceae, algae, mollusks, andmarine organisms, plants, microbial organisms, as well as transgenicorganisms endowed with the enzymatic capacity to form long chain ω-3PUFA. Likewise, additional precursors of known SPMs and novel SPMs maybe identified and enriched in such oils. In addition, the SPMs and SPMprecursors may be present as esters and amides. The esters can benatural esters such as triglycerides, diglycerides, monoglycerides, andphospholipids, as well as esters prepared during the industrialprocesses commonly employed in the fish oil industry permitting theconcentration of EPA and DHA from crude and refined fish oils, inparticular the form of ethyl esters.

Any SPM, SPM precursor, or mixtures of SPMs and SPM precursors that arefound in oils obtained from long chain ω-3 PUFA-containing organisms canbe enriched or concentrated employing extraction and separation methods,for example, distillation technologies, and chromatographicfractionation and separation technologies.

The present invention has discovered that, and unanticipated to theknown state of the art, SPMs and SPM precursors can be found assaponifiable substances in crude oils, in subsequently derived refinedoils, and in oils in which the levels of long chain ω-3 PUFA, such asEPA and DHA, have been concentrated in the form of ethyl esters. Forexample, it is shown in Example 2 (FIG. 5A) that a widely employed crudeoil extracted from anchovy, which is considered a good starting materialfor the omega-3 industry since it is relatively rich in EPA and DHA,contains the D-series resolvins RvD1 and RvD2 (both in acylated form).The saponifiable form of the SPMs or SPM precursors can also be presentas ethyl esters as a result of the transesterification of long chain ω-3PUFA-containing oils with ethanol to obtain fatty acid-ethyl ester oilsthat can be concentrated and fractionated employing specificdistillation, extraction and chromatographic industrial proceduresemployed for ω-3 PUFA-ethyl ester concentration and purification. Forexample, many monohydroxylated lipid mediators which can function as SPMprecursors are found in saponifiable form in the ethyl esterifiedomega-3 concentrates manufactured from anchovy oil, tuna liver oil, andin ethyl ester omega-3 concentrate manufactured from a mixture ofmollusks and fish (FIG. 2, 3A, FIGS. 3B, 5C, 6A, and 6B). The presenceof the esterified forms of SPM precursors and SPMs themselves present inethyl ester concentrates of long chain ω-3 PUFA oils demonstrates thatthese SPM precursors and SPMs were originally present in acylated formin the crude marine oils and organisms from which the crude oil wasextracted. In the process of transesterification of a refined oil withethanol, these acylated SPM precursors and SPMs also becometransesterified to the corresponding ethyl esters. This finding is ofhighly significant and unanticipated nature, since SPMs and SPMprecursors are not known to be found in acylated form in the cells andtissues of organisms which are used for the preparation of ω-3PUFA-containing oils manufactured for use as e.g. nutritionalsupplements and pharmaceutical ingredients. This aspect of the inventiondoes not exclude the presence or enrichment in long chain ω-3PUFA-containing oils of SPMs and SPM precursors as free carboxylicacids, which are the chemical form of SPMs and SPM precursors previouslydescribed in the literature to be formed within cells and organisms fromlong chain ω-3 PUFA substrates. In addition, the oils containing SPMs orSPM precursors can contain long chain ω-3 PUFA, such as EPA and DHA.

Another aspect of the invention is a method for the production of oilswith anti-inflammatory or resolution-stimulating activity and containingmeasurable levels of SPMs and/or SPM precursors. The method includes thefollowing steps; i) measuring the presence or concentration of SPMs orSPM precursors in a long chain ω-3 PUFA-containing oil. This can be e.g.a crude, refined, or concentrated long chain ω-3 PUFA-containing oil;ii) fractionating the oil into a plurality of fractions; iii) measuringthe anti-inflammatory or resolution-stimulating activity of thefractions; iv) and, optionally, repeating the three steps, in order toobtain an oil with anti-inflammatory or resolution-stimulating activity,and containing or enriched with at least one SPM or SPM precursor.

Measuring the presence of SPMs and SPM precursors in an oil permitsassessing or gauging the suitability of an oil to be fractionated inorder to obtain an oil which contains at least one SPM or SPM precursor,has a desirable combination of SPMs and SPM precursors, or which has anenrichment with at least one SPM or SPM precursor. The presence andabsolute levels of SPMs and SPM precursors in a given sample or fractioncan be determined by analytical chemistry techniques such as liquidchromatography coupled to electrospray ionization tandem massspectrometry (LC/ESI-MS/MS), and gas chromatography/mass spectrometry(GC/MS) (Yang, 2011). Other techniques for detecting and/or quantifyingSPMs and SPM precursors that might be used include immunoassays such asthe Resolvin D1 ELISA assay marketed by Cayman Chemical Company (AnnArbor, Mich.), and Neogen Corporation's LXA4 and AT-LXA4 assay kits.

Fractionating a crude, refined, or concentrated long chain ω-3PUFA-containing oil into a plurality of fractions, permits theproduction of oils which contain higher concentrations of the at leastone SPM or SPM precursor than other fractions, or contain a desiredcombination of SPMs or SPM precursors. The fractionation of oils can beachieved with separation and extraction methods. Because the SPMs and/orSPM precursors present in the oils from natural sources will differaccording to the natural source from which the crude oil was obtained,different methodologies will lead to various compositions of SPMs andSPM precursors in the various oils employed for finished productpreparation.

Several extraction and separation technologies are available to obtainoils containing or enriched in at least one SPM or SPM precursor. Suchtechnologies can operate on the molecular form in which the SPMs and/orSPM precursors were isolated, such as triglycerides in fish andvegetable oils, or phospholipids and triglycerides present in krilloils, or after transformation into a different chemical form, notablyfatty acid ethyl esters. Oils composed of fatty acid ethyl esters cansubsequently be employed to manufacture remodeled triglycerides orcompositions containing high levels of free fatty acids. Oils containingSPMs and/or SPM precursors can be obtained from the here mentioned longchain ω-3 PUFA-containing crude oils as starting materials by one or acombination of several technologies. Suitable methodologies will beexplained hereafter.

The extraction process involves heating the raw material containing longchain ω-3 PUFA (e.g., fish, krill, squid, or algae) to temperatures upto 95° C. The heat treatment step yields a “pre-pressing” liquidcontaining both water and fat. Subsequent pressing (of the left-oversolid material obtained in the thermal treatment) at pressures of 130 to170 bar and concomitant pressing with a screw-press yields a pressingliquid. The pre-pressing liquid and pressing liquid can be combined(“press water”) and then fed into a 2-phase decanter to remove solidsand obtain clarified “press water.” The press-water is “de-oiled” bycentrifugation in a separator, yielding a turbid oil. The turbid oil canthen be “polished” by means of an additional centrifugation step with aseparator to obtain a “crude” oil. An alternative process employs atwo-phase decanter instead of a screw-press which simplifies the processby directly separating solid from oil-containing fluid, from which theoil is separated by an oil separator (polishing). In a third process, adecanter is used to separate heat-coagulated raw material directly intosolid, water, and oil. The oil can then be polished with a separator toremove traces of water. Temperatures during separation processes aremaintained between 95° C. and 98° C. Preferably, the application of heatis limited to the shortest time required to separate fat fromheat-coagulated protein and water. Most of the SPMs and/or SPMprecursors in the crude oil obtained by any of these extraction methodsare in acylated form as esters within glycerides and phospholipids, andas amides.

A crude oil can be cleaned by a chemical “refining” process. This stepinvolves washing the oil with alkaline and acid solutions in order toneutralize the oil, separation with a separator to remove the aqueouswash from the oil, hot water washing, a “bleaching” treatment of the oilwith diatomaceous earths, activated carbon or silica, followed byfiltering in order to remove (such as colored carotenoids, metals,contaminants) impurities by adsorption, and vacuum drying. Generally,temperatures between 95° C. and 98° C. are maintained during refiningprocesses. An additional deodorization step can be applied whichinvolves heating the oil up to 200° C. to remove volatile substances.

Alternatively, cold extraction techniques might be used to obtain oilsthat contain SPMs and/or SPM precursors.

Winterization of an oil is a process by which the oil is cooled at acontrolled rate permitting the differential crystallization of distinctlipids based on differences in melting points—permitting separation ofdifferent lipid classes. This separation technique may be useful in theseparation of waxes and lipids rich in saturated fatty acids from alipid (usually triglyceride) fraction containing a higher content inSPMs and/or SPM precursors or acylated forms thereof.

One or more molecular distillation techniques might also be used forthis purpose. Molecular distillation methods include thin filmdistillation, wiped film distillation, and short path distillation. Inthin film evaporation and distillation, a film of the oil is created byrotating fans or rollers within a closed vessel. By the combinedapplication of low pressure conditions and heating, the differentialevaporation of distinct lipid components is achieved, permitting therelative enrichment of a lipid fraction of interest (i.e., thosefractions containing higher levels of SPMs and/or SPM precursors). Inwiped-film distillation the oil is actively wiped into a film onto aheated surface by a rotating barrel.

Short path evaporation distillation is a molecular distillationtechnique which is particularly useful for fractionation of compoundssensitive to oxidation by air through the introduction of an internalcondenser within the vessel where evaporation or distillation is takingplace. Like with thin film evaporation and distillation thefractionation is carried out under reduced pressure and heating.Pressure losses are diminished in this configuration and lower orshorter heating times may be achieved by this technique. A short pathdistillation plant comprises a supply tank, an evaporator, a vacuumpump, a degasser, rollers, heat exchangers, a condenser, a thermalconditioned tank and a continuous and closed circuit.

Molecular distillation steps can be performed in sequential order toconcentrate a range of structurally similar fatty acids from an oil toobtain an oil fraction of interest. Another technique complementary tomolecular distillation is vacuum rectification, which incorporates anexternal reflux process permitting higher levels of concentration at theinconvenience of higher contact times. Fatty acids in oils can befurther concentrated by means of a selective precipitation step throughthe addition of urea, which selectively complexes saturated andmonounsaturated fatty acids. Additional concentration technologiesencompass ionic exchange employing cation- and anion-exchanging resins.Another technology which can permit selective concentration based onmolecular size and weight is ultrafiltration.

An extraction technology which is of particular usefulness for theextraction of SPMs and SPM precursors is supercritical fluid extraction(SFE). A supercritical fluid extraction plant comprises a supply tank,pumps, a solvent tank, a continuous and closed circuit, an extractioncolumn, atmospheric tanks and separators. By attaining specificcombination of pressure and temperature the mobile phase can be broughtabove its supercritical point. SFE is commonly employed undercountercurrent conditions whereby a steady state is achieved permittingselective enrichment of a component eluting from the top or bottom ofthe extraction column. SFE permits selective enrichment. SFE thuspermits manufacturing of oils which are suitable starting material forsubsequent separation technologies employed for selectively separatingand purifying individual fatty acids, for example as their correspondingethyl ester, permitting concentration up to levels that can approachnear purity.

Chromatographic techniques are useful for achieving significant levelsof separation of individual ethyl-esterified fatty acids, and aresuitable for obtaining oils which are selectively enriched with SPMs andSPM precursors. These include conventional chromatography byhigh-pressure operation, moving-belt chromatography, counter-currentchromatography, and supercritical fluid chromatography (SFC). Ahigh-pressure chromatography employs mixtures of aqueous and organicsolvents pumped at elevated pressure through a column containing astationary phase. The stationary phase can have different polarities andparticles size and geometries. By choosing optimal combinations ofmobile phase, stationary phase, temperature acceptable separation offatty acid-ethyl esters can be achieved.

Supercritical fluid chromatography (SFC) employs supercritical fluid(usually carbon dioxide) as a solvent and mobile phase. By carefulmodulation of the supercritical fluid density through pressure andtemperature, eluting conditions can be optimized for the separation ofindividual lipids within a sample. The advantage of this technique isthe employment of near ambient temperatures and the exclusion of oxygenduring the chromatographic procedure to eliminate the risk forinadvertent oxidation. Installations encompass a supply vessel, pumps, amobile phase tank, a continuous and closed circuit, a chromatographycolumn, atmospheric tanks and separators. SFC permits chromatographicseparation of fatty acid-ethyl esters. The mobile phase, since it is agas at ambient pressure and temperature, is easily removed from thefinal oil fraction.

Preferred techniques for obtaining SPMs and/or SPM precursor-containingoils by fractionation of long chain ω-3 PUFA-containing oils aresupercritical fluid extraction (SFE) and supercritical fluidchromatography (SFC). These techniques may be complemented optionally byone or more additional fractionation steps allowing enrichment of one ormore defined SPMs and/or SPM precursors. Examples are described below.The following ranges of SFE and SFC conditions can be employed:temperature range between 27-60° C., pressure range between 80-180 bar,with silica, modified silica, reversed phase, chiral and argentatedstationary phases, and solvent/feed ratios of 10-800 (Kg/Kg).

The combination of SFE and SFC permits enrichment of one or severalspecific SPMs and/or SPM precursors. The capacity to separate SPMsand/or SPM precursors furthermore permits recombining specific oilfractions in order to obtain a versatile range of ratios andcombinations.

Additional chromatographic steps can be performed employing veryspecialized enrichment technologies such as chiral separations, andmetal-affinity chromatography such as argentation chromatography withimmobilized silver salts.

As a result of the technology employed for the preparation of the oilscontaining or enriched for SPMs and/or SPM precursors, the chemicalforms of these molecules is commonly one of the following; ethyl esterswhen present in omega-3 concentrates, acylated within glycerides andphospholipids typical for crude and refined oils, or found as freecarboxylic acids dissolved within the oils. Other chemical forms of theSPMs and SPM precursors may be found in crude and refined oils, such asamides. In a further embodiment, the SPMs and SPM precursor moleculescan be firther transformed according to known methods. For example,SPM-ethyl ester-containing oils can be transesterified again (eitherchemically or enzymatically) with a triglyceride or phospholipid to forma remodeled triglyceride or phospholipid, respectively. Esterified SPMsand SPM precursors can also be hydrolyzed to obtain the correspondingfree fatty acid form, as a salt or the conjugate acid.

In a particular embodiment this invention furthermore may ultimatelypermit naturally-occurring SPMs and/or SPM precursors to be purified tohomogeneity or near homogeneity (e.g., more than 80, 90, 95, 96, 97, 98,or 99% pure by weight).

Furthermore, crude oils originating from the same or from differentorganisms containing long chain ω-3 PUFA can be combined and used asstarting material for subsequent enrichment procedures to obtain SPMsand SPM precursor-containing oils.

Determination of the anti-inflammatory or resolution-stimulatingactivity of the fractions containing higher concentrations of the atleast one SPM or SPM precursor than other fractions, or containing adesired combination of SPM or SPM precursors, will establish theusefulness of the oil to make a therapeutic anti-inflammatory orresolution-stimulating composition. This can be preferably performed invivo, in experimental models of inflammation that permit assessment ofanti-inflammatory efficacy and potency, or measuring theresolution-activating activity of the oil fraction (Bannenberg, 2005).In vitro and cellular models may be employed for this purpose in orderto measure a particular cellular or molecular aspect ofanti-inflammatory or proresolving activities on the in vivo inflammatoryresponse.

Another aspect of the invention is that the purposefully manufacturingof SPM- and SPM-precursor-containing or -enriched oils withanti-inflammatory and resolution-enhancing activities, can be used forreducing inflammation or stimulating the resolution of inflammation in asubject, the method comprising the step of administering an effectiveamount of an oil. The oils can be used for treating inflammation ordiseases associated with inflammation, or preventing inflammation ordiseases associated with inflammation. Fractionation of oils permitsobtaining oils with specific anti-inflammatory or resolution-stimulatingactivity. A functional differentiation can be achieved by fractionation,with some oil fractions having anti-inflammatory activity and/orresolution-stimulating (proresolving) activity, other oil fractionshaving no significant anti-inflammatory activity, and/or even oilfractions may be obtained having an inflammation-promoting activity.Specific oils obtained through fractionation thus have the capability tomodulate the inflammatory response distinctly.

Specific oils and oil fractions containing natural SPMs, SPM precursors,or mixtures of SPMs and/or SPM precursors can be particularly wellsuited to treat or prevent a specific inflammatory condition. Forexample, an oil containing or enriched with a particular SPMs and/or SPMprecursors, or combination of more than one SPM or SPM precursor, mightbe selected for treating rheumatoid arthritis, based on research showingthat these particular molecules are more beneficial for treatingrheumatoid arthritis than either currently used ω-3 PUFA-containingoils, or other SPMs, SPM precursors, or mixtures of SPMs and/or SPMprecursors, or known anti-inflammatory drugs. Other SPMs and/or SPMprecursor-containing oils might be selected for making a composition fortreating a different inflammatory condition, e.g., asthma, based onresearch. This method includes the step of administering to the subjectan effective amount of an oil containing or enriched for at least oneSPM or SPM precursor and having anti-inflammatory orresolution-stimulating activity.

The SPMs and/or SPM precursor-containing oils might further comprise acarrier or an excipient.

As used in the present invention, the terms “subject” or “patient”refers to animals, including mammals, preferably humans.

As used in the present invention, the terms “administer”,“administering” or “administration”, as used herein, refer to directlyadministering an oil or oil-containing composition to a subject orpatient, which will deliver an effective amount of the active compoundor substance to the subject's or patient's body.

As used in the present invention, the term an “effective amount” or “anamount effective to” means an amount adequate to cure or at leastpartially ameliorate the symptoms of a condition, disease or itscomplications.

Another aspect of the invention encompasses the anti-inflammatory orresolution-stimulating oils containing SPMs or SPM precursors in thatthese oils also contain long chain ω-3 PUFA. These can be EPA, and DHA,but also other long chain ω-3 PUFA such as stearidonic acid ordocosapentaenoic acid.

Another aspect of the invention relates to the making of nutritionalsupplements, pharmaceutical formulations, and cosmetic formulationscomprising an effective amount of SPM- and SPM-precursor-containing or-enriched oils with anti-inflammatory or resolution-enhancing activityobtained from organisms containing long chain ω-3 PUFA. After obtainingan oil or oil fraction which contains or is enriched for one or morenaturally present SPMs and/or SPM precursors and that hasanti-inflammatory or resolution-stimulating activity, the oil can beused to make a nutritional supplement, a pharmaceutical formulation, ora cosmetic formulation.

As used in the present invention, the term “Pharmaceutically acceptable”refers to those compounds, materials, compositions, supplements,formulations, and/or dosage forms which are, within the scope of soundmedical judgment, suitable for contact with the tissues of human beingsand animals without excessive toxicity, irritation, allergic response,or other problem complications commensurate with a reasonablebenefit/risk ratio.

In addition to the oil containing SPMs and/or SPM precursors, thenutritional supplements and pharmaceutical and cosmetic formulationsmight contain other ingredients. For example, in preferred embodimentsthe SPM and/or SPM precursor containing oils are mixed, dissolved,emulsified (e.g., in oil/water, water/oil, or double emulsions), orsuspended in a matrix or base. The matrix or base can, e.g., be anedible oil such as ω-3 PUFA-containing oils, an ω-3 PUFA concentratecontaining high levels of EPA, or DHA, or mixtures of EPA and DHA, oranother edible oil suitable for consumption or administration. Thematrix or base might also be water or an aqueous buffer. The oilscontaining SPMs and/or SPM precursors might also be prepared inliposomes, nanoparticles, or microparticles.

To enhance shelf life, the supplements and formulations might alsocontain one or more stabilizers including antioxidants such as one orseveral tocopherols, ascorbic acid and ascorbyl-fatty acid derivatives,and other antioxidants which are commonly used in the stabilization ofdietary oils, such as rosemary extract. The oils might furthermore bepackaged in containers that minimize exposure to oxygen, heat, andincident light. These conditions will specifically augment the stabilityof the SPMs and SPM precursors by preventing or limiting oxidation andisomerization of double bonds. Stability of the bulk oil or theformulated oil will also benefit from these conditions since the SPMsand SPM precursors are dissolved in oils with a significant level ofPUFA that are sensitive to oxidation.

The supplements and formulations might also include one or more activeingredients such as aspirin, other non-steroidal anti-inflammatorydrugs, vitamins, anti-oxidants, flavonoids, minerals, trace elements,fatty acids, lycopene, S-adenosylmethionine, oleocanthal, resveratrol,pterostilbene, bioactive proteins and peptides such as bromelain,oligosaccharides, glucosinolates, and plant extracts such as Boswelliaserrata, mangosteen, capsicum, turmeric, ginger, tea, neem, and/orwillow bark extract. Ingredients are not limited to the here mentionedexamples.

Specific nutritional supplements can be made to support specific healthconditions that include a fish oil, a krill oil, or a long-chain ω-3PUFA concentrate supplemented with an oil containing SPMs or SPMprecursors, together with glucosamine and chondroitin for arthritis, orwith zinc, lutein and zeaxanthin for eye health.

Other nutritional supplements containing oils with SPMs and SPMprecursors are multi-vitamin preparations, sports nutrition, fortifiedfish oil capsules, oral healthcare products such as tooth paste andmouthwash, and specific oils used as food such as spreads, dressings,cooking oils, snacks, nutritional drinks, soft gels, chewing gums, andin infant formulas.

The oils described herein might be included along with one or morepharmaceutically acceptable carriers or excipients to makepharmaceutical formulations which can be administered by a variety ofroutes including oral, rectal, vaginal, topical, transdermal,sublingual, subcutaneous, intravenous, intramuscular, insufflation,intrathecal, and intranasal administration. Suitable formulations foruse in the present invention are found in Remington's PharmaceuticalSciences, Mack Publishing Company, Philadelphia, Pa., 17th ed. (1985).

The active ingredient(s) can be mixed with an excipient, diluted by anexcipient, and/or enclosed within a carrier which can be in the form ofa capsule, sachet, paper or other container. When the excipient servesas a diluent, it can be a solid, semi-solid, or liquid material, whichacts as a vehicle, carrier or medium for the active ingredient. Theformulations can be in the form of tablets, pills, powders, lozenges,sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups,aerosols (as a solid or in a liquid medium), ointments, soft and hardgelatin capsules, suppositories, sterile injectable solutions, sterileliquids for intranasal administration (e.g., a spraying device), orsterile packaged powders. The formulations can additionally include:lubricating agents such as talc, magnesium stearate, and mineral oil;wetting agents; emulsifying and suspending agents; preserving agentssuch as methyl- and propylhydroxy-benzoates; sweetening agents; andflavoring agents. The supplements and formulations of the invention canbe formulated so as to provide rapid, sustained or delayed release ofthe active ingredients alter administration to the patient by employingprocedures known in the art.

For preparing solid formulations such as tablets, the oil is mixed witha pharmaceutical excipient to form a solid preformulation compositioncontaining a homogeneous mixture of a compound. Tablets or pills may becoated or otherwise compounded to provide a dosage form affording theadvantage of prolonged action. For example, the tablet or pill cancomprise an inner dosage and an outer dosage component, the latter beingin the form of an envelope over the former. The two components can beseparated by an enteric layer which serves to resist disintegration inthe stomach and permit the inner component to pass intact into theduodenum or to be delayed in release. A variety of materials can be usedfor such enteric layers or coatings, such materials including a numberof polymeric acids and mixtures of polymeric acids with such materialsas shellac, acetyl alcohol, and cellulose acetate.

Liquid forms of the formulations include suspensions and emulsions. Theformulations may be encapsulated, prepared as a colloid, introduced intothe lumen of liposomes, or incorporated in the layers of liposomes. Aliquid formulation may also consist of the oil itself, which may beencapsulated.

The oils are preferably formulated in a unit dosage form of the activeoil and its ingredient(s). The amount administered to the subject orpatient will vary depending upon what is being administered, the purposeof the administration, such as prophylaxis or therapy, the state of thesubject or patient, the manner of administration, and the like all ofwhich are within the skill of qualified physicians, dieticians, andpharmacists. In therapeutic applications, formulations are administeredto a patient already suffering from a disease in an amount sufficient tocure or at least partially arrest the symptoms of the disease and itscomplications. Amounts effective for this use will depend on the diseasecondition being treated as well as by the judgment of the attendingclinician depending upon factors such as the severity of the symptoms,the age, weight and general condition of the patient, and the like.

Specific pharmaceutical formulations could be encapsulated oils to betaken orally for the treatment of diseases with an inflammatorycomponent, sustained release formulas, topical formulations fortreatment of acne, psoriasis, eczema, rosacea, etc., intravenousformulations based on emulsified oils which are useful as clinicalnutrition and parenteral drugs, liposomal preparations, and ti ssue-targeted delivery system s, inhalation formulations, andformulations which can be injected into the central nervous system.

A further embodiment is the formulation of oils containing SPMs and SPMprecursors and having anti-inflammatory or resolution-stimulatingactivity as cosmetics, beauty products, and nutritional cosmetics. Theseformulations include make-up, skin moisturizers, and specific topicalcreams such as sunburn and tanning ointments. In particular, the oilsbeing anti-inflammatory and resolution-stimulating and containing SPMsand SPM precursors, might constitute cosmetics which counteractirritation and inflammation at the site of application.

Methods of Use

The invention features methods for treating a subject (e.g., a humanbeing, dog, cat, horse, cow, goat, pig, fish, and other animals) havinginflammatory condition or a disease with an inflammatory component byadministering to the subject one or more of the oils, supplements, andformulations described herein, in an amount and dosage scheduleeffective to cure, treat, and/or reduce inflammation in a subject. Thetherapeutic use of the SPM and/or SPM precursor-containing oils will beprimarily directed to treat or prevent any of many possible ailments,disorders, and diseases that include an aspect of inflammation in theiretiology or symptoms. The use may furthermore encompass conditions anddiseases that have been reported to be ameliorated by increasedingestion of EPA/DHA or fish oils (e.g., hypertriglyceridemia,arrhythmias, or depression). Examples of inflammatory conditions includecardiovascular disease (e.g., atherosclerosis, high blood pressure,hypercholesteremia, hypertriglyceridemia, endothelial hyporeactivity,cardiac infarction, cerebral stroke), aspects of metabolic syndrome(e.g, loss of insulin sensitivity, obesity, hepatic steatosis,cholestasis), neurodegenerative diseases (Alzheimer's disease, Parkinsondisease, multiple sclerosis, apraxia), atopic/allergic reactions,cancer, osteoarthritis, rheumatoid arthritis, inflammatory pain, acne,psoriasis, rosacea, asthma, acute lung injury, chronic obstructivepulmonary disease, cystic fibrosis, sepsis, allergic rhinitis,sinusitis, periodontitis, inflammatory bowel disease, Crohn's disease,macular degeneration, dry eye syndrome, gastric ulceration, cancer, andauto-inflammatory disorders. The oils described herein may also besuitable for treating distinct forms of acute and chronic pain andhypersensitivity to physical and chemical stimuli. The oils describedherein might also be useful for treating conditions caused by thedysregulation of angiogenesis, platelet aggregation and coagulation,bone growth, tissue healing, blood pressure regulation, haematopoiesis,and lipid homeostasis. The oils described herein might also be usefulfor lowering the macroscopic and physical signs of inflammation such asswelling, edema, redness, fever, pain, and inflammatory sickness.

The oils described herein, since they contain long chain (ω-3PUFA-derived lipid mediators with anti-inflammatory and pro-resolvingactivity, may furthermore obviate the need to augment tissue levels oflong chain ω-3 PUFA from which these substances may be formed within asubject's body after dietary supplementation.

The oils described herein might also be administered to subjects havingincreased or abnormal levels of inflammatory markers such ashigh-sensitivity C-reactive protein (hs-CRP), serum amyloid A,erythrocyte sedimentation rate, soluble adhesion molecules (e.g.,E-selectin, P-selectin, intracellular adhesion molecule-1, vascular celladhesion molecule-1), cytokines (e.g., interleukin-1β, -6, -8, and -10and tumor necrosis factor-α), fibrinogen, and/or activated white bloodcells (e.g., leukocytes with enhanced rates of production of reducedoxygen and nitrogen species; non-spherical neutrophils, and monocyteswith increased vacuolization). In this regard, supplements andformulations of the inventions might be used to reduce the levels of oneor more of these inflammatory markers by at least 99, 95, 90, 80, 70,60, or 50%; or to reduce these levels to within ranges considerednormal.

The supplements and formulations may also be administered to a subjectto prevent inflammation.

EXAMPLES Example 1: Fractionation of an Omega-3 PUFA Ethyl Ester Oil byIndustrial-Scale Supercritical Fluid Chromatography Permits Enrichmentof Esterified Precursors of SPMs and Manufacturing of Distinct OilFractions with Different Anti-Inflammatory Activity

To evaluate the anti-inflammatory activity of distinct fatty acid-ethylester oil fractions obtained during supercritical fluid chromatography(SFC) for the industrial-scale manufacturing of EPA-ethyl ester andDHA-ethyl ester concentrates, a murine model of subcutaneous sterileinflammation was established which permitted measuring the effects oforally administered oil fractions on the pro-inflammatory phase of theinflammatory response. Eight consecutively-eluted oil fractions wereproduced by SFC at industrial scale by fractionation of an intermediatelong chain ω-3 fatty acid-ethyl ester concentrate containing 70% EPA-EEand DHA-EE combined, which upon its turn had been obtained byindustrial-scale supercritical fluid extraction (SFE). SFC fractionationis carried out in the following way. A raw material tank, previouslyblanketed with nitrogen, is charged with the long chain ω-3 PUFA ethylester concentrate. The tank content is warmed up if necessary andtemperature stabilized approximately between 20-40° C. The oil isprocessed batch-wise by passing it through a chromatographic column. Oilvolumes weighing between 7.5 and 9.5 kg are pumped adjusting pressureand temperature at about 110-135 bars and 20-40° C. Carbon dioxide ispumped at the same time at 110-130 bars and between 43.5-45.5° C. Bothflows (omega-3 concentrate and carbon dioxide) are injected onto thehead of the chromatographic column flowing inside at a pressure between98-102 bar and a temperature between 43.5-45.5° C. Taking advantage ofdifferences in retention of the components which make up the oil to beseparated through the chromatographic column, filled with modifiedsilica stationary phase, different fractions are collected. Totalelution times of single fractionation runs are between 40-85 minutes.Eluted material is collected in consecutively-eluted fractions that lastbetween 2-20 minutes. The ratio between mobile phase (supercriticalcarbon dioxide) and feed (omega-3 concentrate) is between 600-850 Kgsolvent/Kg feed.

In order to initiate inflammation, Escherichia coli lipopolysaccharide(LPS; serotype 127:B8, purified by trichloroacetic acid extraction,Sigma-Aldrich) was injected sub-cutaneously as a single dose (5milligram per kilogram in a 200 microliter volume of sterile salive) inthe dorsal hind flank of a mouse (CD1 mice of 9 weeks age and weighingapproximately 30 grams, purchased from the Charles River company).Neutrophil infiltration into the site of inflammation was measurednon-invasively by bioluminescence emitted by conversion of luminol bythe neutrophil enzyme myeloperoxidase (Gross, 2009), permittingassessment of inflammatory changes over a 6 hour time period. Theemployment of a sub-cutaneous model of inflammation permittedreproducible bioluminescence measurements of neutrophil activity inorder to be able to measure statistically significant changes inneutrophil activity upon administration of test substances. Thirtyminutes prior to the administration of LPS, 100 microliter of vehiclecontrol (sterile salive), indomethacin (dose; 10 milligram perkilogram), or one of the eight oil fractions obtained by SFC, wasadministered by gavage, reflecting the oral route (per os, (p.o.)) ofadministration. The non-steroidal anti-inflammatory compoundindomethacin was used as a positive control to confirm that theinflammatory response induced by LPS could be inhibited. FIGS. 1A-H showthe effect of a series of consecutively eluted oil fractions (number1-8, respectively), obtained by industrial-scale SFC fractionation ofthe intermediate long chain ω-3 fatty acid-ethyl ester concentrate(containing 70% EPA-EE and DHA-EE combined), on acute inflammatorychanges occurring subcutaneously in mice induced by subcutaneous (s.c)administration of lipopolysaccharide (LPS). Open circles; inflammationinduced by LPS s.c. (n=40). Open squares; indomethacin 10 mg/kg p.o. 30minutes prior to LPS s.c. (n=6). Open triangles; 100 microliter of eachoil fraction number 1-8 depicted in panel A-H, respectively, eachadministered once by gavage 30 minutes prior to LPS (n=6 per tested oilfraction). Values are mean standard error of the mean. Statisticallysignificant differences (Student's t-test; P<0,05) in inflammation areindicated by: * (oil fractions given before LPS compared to vehiclegiven before LPS), # (indomethacin given before LPS compared to vehiclegiven before LPS), and t (oil fractions given before LPS compared toindomethacin given before LPS).

As shown in FIG. 1, indomethacin inhibited LPS-induced inflammation by26% after 3 hours and 44% after 6 hours (number of independentobservations n=40), in comparison to mice which had received saliveinstead of indomethacin (n=40). Of interest, fractionation by SFC of anethyl ester oil containing high levels of long chain ω-3 PUFA-ethylesters permitted the production of different oil fractions that hadmarkedly distinct activities on inflammation alter oral administration(n=5 for each tested oil fraction). Three oil fractions induced ananti-inflammatory action after oral administration. Oil fraction 1significantly reduced inflammation by 61% three hours after the start ofLPS-induced inflammation, and by 82% at six hours (FIG. 1A). Oilfraction 7 significantly reduced inflammation by 49% after three hours(FIG. 1G). Oil fraction 8 significantly reduced inflammation by 66%alter 90 minutes (FIG. 1H). Oil fractions 2, 3, 4, and 6 did notsignificantly change the LPS-stimulated inflammatory response (FIG. 1,panels B, C, D and F, respectively). Of interest is that theanti-inflammatory actions of several oil fractions had significantlyhigher efficacy than the widely used anti-inflammatory compoundindomethacin itself, namely oil fractions 1 and 8. Furthermore, themarked anti-inflammatory activity of oil fraction 1 also points to aresolution-stimulating activity that, already after 6 hours, hasactively brought back the neutrophilic inflammatory response nearly tothe non-inflamed state. The industrial scale SFC fractionation permittedobtaining sufficient functional differentiation such that one oilfraction, 3, potentiated inflammation at the earliest time point, namelya more than doubling of neutrophil activity at 90 minutes, whereafterthe extent of the neutrophilic response normalized to the responseobserved in vehicle-treated animals. This points out that this oil couldfacilitate a more rapid inflammatory response towards a bacterialinfectious stimulus. In summary, the results demonstrate that throughthe fractionation of a long chain 0-3 PUFA-rich oil it is possible toachieve oil fractions that have distinct activities on the inflammatoryresponse, and that oil fractions are obtained with significantanti-inflammatory activity after oral administration.

The same oil fractions which had been evaluated for theiranti-inflammatory activity were analyzed for their relative or absolutelevels of precursors for SPM biosynthesis as well as the SPM themselves.All oils were stabilized by addition of butylated hydroxytoluene inorder to avoid inadvertent oxidation. Liquid-liquid extractions of theoil fractions to isolate SPMs and their precursors did not reveal thepresence of measurable levels of any mono-, di-, and tri-hydroxylatedderivatives of PUFA, such as EPA, DHA, or AA. However, when oils werehydrolyzed by alkaline hydrolysis (10 M NaOH, stirring, 3 hours, at 20°C.), a significant number of lipid mediators derived from EPA, DHA andAA were detected. FIG. 2 shows the relative abundance of theethyl-esterified and saponifiable forms of various monohydroxylatedderivatives of the polyunsaturated fatty acids EPA and DHA, inconsecutively-eluted oil fractions of an industrial scale SFCfractionation of the intermediate long chain ω-3 fatty acid-ethyl esterconcentrate (containing 70% EPA-EE and DHA-EE combined). The fractionsnumbered 1-8 are the same as those tested for anti-inflammatory activityas shown in FIG. 1. Values are means of duplicate measurements of peakareas of mass spectrometric recordings of ion transitions correspondingto each PUFA derivative. No measurable levels of the corresponding freefatty acid forms of the same PUFA derivatives could be found in theseoil fractions. (Abbreviations; HEPE, hydroxy-eicosapentaenoic acid;HDHA, hydroxy-docosahexaenoic acid). Since these oil fractions arederived from ethyl-esterified oil employed in the industrial scaleconcentration and purification of EPA-EE and DHA-EE, the measured lipidmediators are ethyl esters themselves. Several of the measured compoundsare known as intermediate precursors for the formation of SPMs, such as4-hydroxy-docosahexaenoic acid (4-HDHA;4-hydroxy-5E,7Z,10Z,13Z,16Z,19Z-docosahexaenoic acid), and18-hydroxy-eicosapentaenoic acid (18-HEPE;18S-hydroxy-5Z,8Z,11Z,14Z,16E-eicosapentaenoic acid). 18-HEPE is aprecursor for the formation of E-series resolvins, and 4-HDHA is knownto have anti-inflammatory and tissue-protective actions invasoproliferative retinopathy, and may act as a precursor for4-HDHA-derived SPMs. The various measured SPM precursors distributeddifferentially into the various oil fractions obtained by SFC. Thisobservation indicates that fractionation of commonly employed ω-3PUFA-containing oils can permit the manufacturing of oils containingdefined presence, combinations, and levels of distinct PUFA-derivedlipid mediators.

One derivative of DHA, 17-hydroxy-docosahexaenoic acid (17-HDHA;17-hydroxy-docosa-4Z,7Z,10Z,13Z,15E,19Z-hexaenoic acid), is of interestas a precursor for SPM biosynthesis, namely as a central precursor forthe formation of the D-series resolvins RvD1, RvD2, RvD3 and RvD4 withpotent anti-inflammatory and inflammation resolving bioactivity. FIG. 3Ashows the concentration of the ethyl ester of 17-HDHA in severalconsecutively eluted oil fractions of industrial scale SFC of theintermediate long chain ω-3 fatty acid-ethyl ester concentrate(containing 70% EPA-EE and DHA-EE combined), corresponding to the samefractions as shown in FIGS. 1 and 2. Quantification of 17-HDHA-ethylester as a saponifiable substance in the consecutively eluting SFC oilfractions was carried out using internal standards and LC-triplequadrupole mass spectrometry. Values are mean standard error of the mean(n=3 individual chromatographic separations, measured in duplicate). Theresults show that 17-HDHA-ethyl ester is enriched in the first elutingfraction, reaching concentrations of approximately 110 mg/l (0.01% w/v).No measurable levels of the corresponding free fatty acid form of17-HDHA could be found in these fractions. Measurement of 17-HDHA levelsin the first oil fraction of several SFC-fractionated lots of thisintermediate long chain ω-3 fatty acid-ethyl ester concentrate, which isproduced by industrial-scale supercritical fluid extraction (SFE), hasindicated that the range of concentrations of 17-HDHA in this fractionlies in the range of 30-110 mg/l. This shows that specificindustrial-scale fractionation steps can be devised to enrich specificSPMs and SPM precursors into defined oil fractions.

It was of interest to determine that these SPM precursors found in anω-3 PUFA-rich oil are of natural origin like the PUFA-ethyl estersthemselves in which these substances are dissolved. To that end a chiralhigh performance liquid chromatography-triple quadrupole massspectrometric analysis of the 17-HDHA-ethyl ester found enriched infraction number 1 was carried out in order to determine the relativeabundance of the stereoisomers 17S-HDHA and 17R-HDHA (top panel). Oilfraction number 1 analyzed here is the same as oil fraction number 1shown in FIGS. 1, 2, and 3A. Evaluation of co-migration of the observedlipid mediators with authentic synthetic standards (bottom panel) of thestereoisomers and selected ion monitoring of specific mass transitionsby triple quadrupole mass spectrometry indicate that 17-HDHA-ethyl esterin fraction 1 is the natural S stereoisomer. The bottom panel shows theretention times of authentic synthetic standards of the stereoisomers of17-HDHA, 14-HDHA, 7-HDHA and 4-HDHA. Also 4-HDHA is shown here to bepresent predominantly as the natural S stereoisomer. Chemical oxidationis not responsible for the 17-HDHA and 4-HDHA present in oil fraction 1,since the products are not racemic. Since the S-stereoisomer ofmonohydroxylated PUFA is the naturally formed isomer formed by mostlipoxygenases, the presence of this stereoisomer in this oil fractionindicates that 17-HDHA and 4-HDHA have a natural origin and areco-extracted and co-purified with long chain ω-3 PUFA all along theindustrial process up to the step where SFC fractionation was carriedout. Fractionation by a dedicated separation technology, such assupercritical fluid chromatography shown here, furthermore permits thefractionation of select SPMs and SPM precursors of natural origin intospecific oil fractions.

With respect to 17-HDHA, it is possible that the anti-inflammatoryactivity of oil fraction 1 (shown in FIG. 1A) can therefore beexplained, at least in part, due to the selective enrichment of thisanti-inflammatory SPM precursor into this oil fraction. In order todetermine the anti-inflammatory action and contribution of 17-HDHA, theanti-inflammatory effect of fraction 1 was determined in a well-knownmodel of sterile inflammation. FIG. 4 shows the anti-inflammatory effectof oil fraction number 1 administered by gavage in a murine model ofperitoneal inflammation induced by intraperitoneal administration of theyeast membrane extract zymosan A. Selective changes in specificinflammatory cell populations in the inflammatory exudate 4 hours afterinitiation of inflammation were determined. Vehicle (100 microlitersterile salive), 100 microliter oil fraction 1, or 1 microgram synthetic17S-HDHA (Cayman Chemicals) in sterile salive was administered by gavage30 minutes prior to intraperitoneal injection of 0.1 mg zymosan A. Oilfraction 1 analyzed here is the same as fraction number 1 shown in FIGS.1, 2, and 3. After 4 hours, the inflammatory exudate was recovered andthe changes in the number and types of inflammatory cells determined byfluorescent-activated cell sorting employing specificfluorescently-labeled antibodies. Values are mean standard error of themean of 67 individual mice. Statistically significant differences(Student's t-test) are indicated by *(P<0.05) or # (P<0.10) forcomparisons of inflammatory exudate cell numbers obtained aftertreatment with oil fraction 1 or compared to the vehicle-treated mice.As shown in FIG. 4, administration of oil fraction 1 significantlydecreased the total number of exudate cells and the number ofpolymorphonuclear leukocytes (PMN). This anti-inflammatory effect wasreproduced by the oral administration (gavage) of 17S-HDHA. Nostatistically significant changes were measured for monocytes,macrophages or lymphocytes. The result indicate that theanti-inflammatory efficacy upon oral administration of oil fraction 1containing approximately 100 mg/l (10 microgram in 100 microliter)17S-HDHA-ethyl ester, is very similar to the anti-inflammatory action ofsynthetic 17S-HDHA. The results furthermore show that oil fraction 1 hassystemic anti-inflammatory efficacy after oral administration in twodistinct models of acute inflammation in mice, namely zymosan-initiatedperitonitis and sub-cutaneous inflammation induced bylipopolysaccharide.

Example 2: Presence of SPMs and SPM Precursors in Oils of Natural Origin

FIG. 5A shows the presence of two resolvins, resolvin D1 and resolvinD2, as saponifiable matter in a crude “1812” fish oil obtained fromPeruvian anchovy. This oil is a common raw material which containsapproximately 18% EPA and 12% DHA. Anchovy “1812” (18/12 means an oilcontaining 18% EPA and 12% DHA) oil is the omega-3 fish oil which iscurrently used in largest volumes world-wide for the manufacturing ofrefined fish oils and fish oil concentrates which have increased levelsof the ω-3 PUFA EPA and DHA. This oil is composed predominantly oftriglycerides, indicating that RvD1 and RvD2 are most probably acylatedwithin triglycerides. Alternatively, or in part, these resolvins mayalso be acylated within a diglyceride or monoglyceride, phosphatidicacid, phospholipid, or other ester or amide species present in this oil.No measurable levels of RvD1 or RvD2 in the free carboxylic acid formwere found in this oil. The chromatogram shows that SPMs which are knownto possess extremely potent anti-inflammatory and resolution-stimulatingactivities, are present in long chain ω-3 PUFA containing oils that arewidely used in the industry for the manufacturing of long chain ω-3PUFA-containing oils as nutritional supplements and pharmaceuticalingredients.

A side-by-side comparison of oils obtained from different long chain ω-3PUFA-containing organisms, demonstrates that 17-HDHA is present assaponifiable substance in oils from anchovy, tuna, krill, and algae, asshown in FIG. 5B. Two different crude anchovy oils, which are commonlyemployed as starting material for the preparation of EPA- andDHA-containing fish oils and EPA- and DHA-ethyl ester concentrates, areshown to contain 17-HDHA (18/12 means an oil containing 18% EPA and 12%DHA, 22/08 means an oil containing 22% EPA and 8% DHA). These twoexemplary crude oils contain up to 30% EPA and DHA combined, but it ishere shown that such oils also contain the SPM precursor 17-HDHA.Measurement of 17-HDHA in tuna, krill and algae oils, which are alsowidely used as dietary supplements for their content of EPA and DHA,showed that these oils also contained significant levels of 17-HDHA.Also in these oils, the measured 17-HDHA was present in the form ofsaponifiable substance pointing to the acylated nature of the SPMprecursor. The tuna, krill and algae oils are commercially availableoils. The tuna oil is a tuna liver oil. The algae oil is aDHA-containing algae oil obtained from a dinoflagellate algae, OrderPeridiniida. In particular, these krill and algae oils are extractedfrom organisms which are purposefully caught or cultured for theirrelatively high content of DHA, and are shown here to contain relativelyhigh levels of 17-HDHA when compared to the measured fish oils. Thepresence of this precursor for D-series resolvins demonstrates that oilscan be produced with defined levels of SPMs or SPM precursors, and thatsuch oils can be employed for the manufacturing of oils in which thesecompounds are further enriched.

A qualitative profiling of the presence of a number of SPMs and SPMprecursors in oils obtained from fish, algae and krill, demonstrated thepresence of specific SPMs and SPM precursors in the oils (FIG. 5C). Themeasurement of the various SPMs and SPM precursors was perfomed byliquid chromatography-tandem mass spectrometry employing diagnostictransitions and co-elution with commercially available lipid mediatorstandards. AH compounds detected correspond to saponifiable substancespresent in the oils, and no corresponding compounds in their freecarboxylic acid form were measurable. For example, both EPA- andDHA-derived monohydroxylated SPM precursors and SPMs are present in twofish oils such as a refined “18/12” anchovy oil (containing 18% EPA and12% DHA) and in an ethyl-esterified tuna liver oil. In contrast,DHA-derived lipid mediators predominate in an oil extracted from algaewhich are cultured for their high levels of DHA. A krill oil wasdemonstrated to contain both EPA and DHA-derived monohydroxylated lipidmediators, but the EPA-derived compound appear more abundant than theDHA-derived lipid mediators. This was also reflected in the presence ofthe epoxygenated derivatives, where the DHA-derived SPM19(20)-epoxy-docosapentaenoic acid (19(20)-EpDPE;19(20)-epoxy-docosa-4Z,7Z,10Z,13Z,16Z-pentaenoic acid) was the dominantepoxy-derivative in the algae oil, and the EPA-derived SPM17(18)-epoxy-eicosatetraenoic acid (17(18)-EpETE;17(18-epoxy-eicosa-5Z,8Z,11Z,14Z-tetraenoic acid) and11(12)-epoxy-eicosatetraenoic acid (11(12)-EpETE;11(12)-epoxy-eicosa-5Z,8Z,14Z,17Z-tetraenoic acid) predominating in thekrill oil. Minor components of interest can be identified, such as shownfor the tuna liver oil, where the double hydroxylated DHA-derived lipidmediator 10S,17S-dihydroxy-Docosahexaenoic acid (10S,17S-diHDHA;10S,17S-dihydroxy-docosa-4Z,7Z,11E,13Z,15E,19Z-hexaenoic acid), as wellas 7,17-dihydroxy-docosapentaenoic acid (ω-3) (7,17-diHDPA (ω-3));(7S,17S-dihydroxy-docosa-8E,10Z,13Z,15Z,19Z-pentaenoic acid (ω-3)) werefound to be present. An oxo derivative of docosapentaenoic acid (ω-3),17-keto-docosapentaenoic acid (ω-3) (17-keto-DPA) was also detected inall tested oils. Oils obtained from different long chain ω-3PUFA-containing organisms thus have markedly different composition withrespect to SPMs and SPM precursors. Differentiation of oils obtainedfrom long chain ω-3 PUFA-containing organisms based on the content ofPUFA-derived lipid mediators is thus possible, and constitutes avaluable base on which to decide which oils might be useful for furtherfractionation by separation and extraction methods to obtain oils withdefined presence, combinations of, and enriched levels of one or moreSPMs and SPM precursors.

Example 3: Enrichment of SPMs and SPM Precursors

FIG. 6A shows the selective fractionation of four exemplary oxygenatedlipid mediators derived from EPA, DHA, and docosapentaenoic acid (DPAω-3), into distinct oil fractions. The starting material which wasfractionated by SFC into eight consecutive oil fractions was a fattyacid-ethyl ester oil containing 56% EPA-EE plus DHA-EE combined. Thisoil corresponds to a long chain ω-3 ethyl ester concentrate manufacturedfrom crude oil extracted from a mixture of marine organisms, includingmarine fish (anchovy, sardine, herring, shad, smelt, salmon, tuna, andbonito) and mollusks (squid, octopus, and cuttlefish). SFC fractionationis carried out in the following way. A raw material tank, previouslyblanketed with nitrogen, is charged with the omega-3 fatty acid ethylester concentrate. The tank content is warmed up if necessary andtemperature stabilized approximately between 2040° C. This oil isprocessed batch-wise by passing it through a chromatographic column. Oilvolumes weighing between 9.0 and 12 kg are pumped adjusting pressure andtemperature at about 110-135 bars and 20-40° C. Carbon dioxide is pumpedat the same time at 110-130 bars and between 43.5-45.5° C. Both flows(omega-3 concentrate and carbon dioxide) are injected onto the head ofthe chromatographic column flowing inside at a pressure between 98-102bar and a temperature between 43.5-45.5° C. Taking advantage ofdifferences in retention of the components which make up the oil to beseparated through the chromatographic column, filled with modifiedsilica stationary phase, different fractions are collected. Totalelution times of single fractionation runs are between 40-85 minutes.Eluted material is collected in eight consecutively-eluted fractionsthat last between 2-20 minutes. The ratio between mobile phase(supercritical carbon dioxide) and feed (omega-3 concentrate) is between600-850 Kg solvent/Kg feed.

The consecutively eluted oil fractions contain different levels ofseveral SPMs and SPM precursora (FIG. 6A), as exemplified by12-hydroxy-eicosapentaenoic acid (12-HEPE;12-hydroxy-eicosa-5Z,8Z,10E,14Z,17Z-pentaenoic acid),14-hydroxy-docosahexaenoic acid (14-HDHA;14-hydroxy-docosa-4Z,7Z,10Z,12E,16Z,19Z-hexaenoic acid),19(20)-epoxy-docosapentaenoic acid (19(20)-EpDPE) and17-keto-docosapentaenoic acid (ω-3) (17-keto-DPA (ω-3)). Values aremeans of two samples for each oil fraction from two independentindustrial scale fractionation runs, measured in duplicate. Results areexpressed as percent enrichment compared to the oil which wasfractionated. The EPA-derived monohydroxylated lipid mediator 12-HEPEwas found to be predominantly present in the second fraction (FIG. 6A).The DHA-derived SPM precursor 14-HDHA was predominantly found in thefirst fraction. 14-HDHA can be further oxygenated to form, for example,14,21-dihydroxy-docosahexaenoic acid which has known potent woundhealing activity. 14-HDHA can also be further oxygenated to7S,14S-dihydroxy-docosahexaenoic acid(7S,14S-dihydroxy-docosa-4Z,8E,10Z,12E,16Z,19Z-hexaenoic acid) which hasanti-inflammatory activity in neutrophilic inflammation. Theepoxygenated lipid mediator 19(20)-EpDPE, which is a cytochrome P450derivative of DHA with anti-inflammatory properties, was foundselectively enriched in later eluting fractions, especially in fraction5. An oxo derivative of docosapentaenoic acid (ω-3), 17-keto-DPA (ω-3),was found to be enriched in fractions 3 to 7. The results indicate thatenrichment of one or more specific oxygenated derivatives of EPA, DHA,and DPA (ω-3) can be achieved by SFC fractionation of long chain ω-3PUFA ethyl ester concentrates. The compounds correspond to saponifiablematerial, and no corresponding free carboxylic acids were measurable inthe oil fractions.

FIG. 6B shows the further enrichment of 17-HDHA-ethyl ester when thefirst SFC fraction of an intermediate long chain ω-3 fatty acid-ethylester concentrate containing 70% EPA-EE and DHA-EE combined isfractionated further. The subfractionated oil corresponds to oilfraction 1 shown in FIGS. 1-3, and which had previously been found tocontain enriched levels of the D-series resolvin precursor 17S-HDHA inthe form of ethyl ester (FIG. 3A). Further fractionation by SFC intothree subfractions eluting between O and 2 minutes (fraction IA), from 2to 7 minutes (fraction 1B), and from 7 to 12 minutes (fraction 1C),afforded additional enrichment into fraction 1B. Values are the relativelevels of 17-HDHA-ethyl ester (means S.D.) in the three sub-fractions.The results indicate that further enrichment of a specific SPM precursorcan be achieved by employing specific separation methods such as SFC.

Example 4: Resolution-Stimulating Activity of an Oil Containing EnrichedLevel of an SPM Precursor

Referring to FIG. 7 which shows the resolution of inflammationstimulated by a SPM precursor-containing oil. The resolution-stimulating(pro-resolving) activity of oil fraction 1 was determined as an exampleto the capacity of an oil fraction containing an SPM precursor, toactivate the resolution of inflammation. FIG. 7 shows the results of anevaluation where the changes in inflammatory cell numbers were measuredby histochemistry of subcutaneous fibrin clots formed during theinflammatory response initiated by subcutaneous administration of LPS.Oil fraction 1 or vehicle (sterile salive) in 100 microliter volume wasadministered by gavage to mice 30 minutes prior to initiation ofinflammation by subcutaneous administration of LPS. LPS-inducedinflammation employed here was the same model as explained in Example 1.Oil fraction 1 is the first eluting fraction of an industrial-scale SFCfractionation of an intermediate long chain ω-3 PUFA-ethyl esterconcentrate containing 70% EPA-EE and DHA-EE combined, and is the samefraction tested in Examples 1-3. This oil fraction 1 corresponds tofraction 1 shown in FIGS. 1-3 and was previously found to containenriched levels of the D-series resolvin precursor 17S-HDHA in the formof ethyl ester. Subcutaneous fibrin clots were isolated at differenttime points (3, 6, 24 and 48 hours) during the inflammatory response,and fixated with 4% formaldehyde for 24 hours at 4° C. Glass slides formicroscopy with 4 micrometer thick paraffin sections were prepared aftertissue dehydration, and stained in modified Wright-Giemsa. Inflammatorycells were counted by microscopy at 400× magnification in full ocularfields of two parts of at least 3 tissue sections per condition. Valuesare average total inflammatory cell counts per ocular field (mean S.D.)of 3 individual mice per time point. Inflammatory cell infiltration incontrol mice reached maximum at 24 hours after administration of LPS,and thereafter inflammation resolved spontaneously towards 48 hours. Inmice which had received oil fraction 1 by gavage, the sub-cutaneousinflammation induced by LPS is almost completely resolved (FIG. 7). TheSPM-precursor enriched oil fraction, which had already been shown tohave a significant anti-inflammatory action on the early neutrophilicpro-inflammatory phase of the inflammatory response (FIG. 1, panel A),is here shown to also have a marked resolution-stimulating(proresolving) activity upon oral administration.

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

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We claim:
 1. A method of reducing inflammation or stimulating resolutionof inflammation in a subject in need thereof, the method comprisingadministering to the subject an effective amount of a compositioncomprising 18-hydroxy-eicosapentaenoic acid (18-HEPE),17-hydroxy-docosahexaenoic acid (17-HDHA), 14-hydroxy-docosahexaenoicacid (14-HDHA), 10-hydroxy-docosahexaenoic acid (10-HDHA), and4-hydroxy-docosahexaenoic acid (4-HDHA), wherein 17-HDHA is present inan amount of at least 100 mg/L.
 2. The method of claim 1, wherein thecomposition further comprises eicosapentaenoic acid (EPA) and/ordocosahexaenoic acid (DHA).
 3. The method of claim 1, wherein 18-HEPE,17-HDHA, 14-HDHA, 10-HDHA, and 4-HDHA are in a form selected from thegroup consisting of a free fatty-acid, an ester, a phospholipid, amono-glyceride, a di-glyceride, a tri-glyceride, a remodeledtri-glyceride, and combinations thereof.
 4. The method of claim 1,wherein 18-HEPE, 17-HDHA, 14-HDHA, 10-HDHA, and 4-HDHA are in are in aform selected from the group consisting of an ethyl ester, a remodeledtri-glyceride, and combinations thereof.
 5. The method of claim 4,wherein 18-HEPE, 17-HDHA, 14-HDHA, 10-HDHA, and 4-HDHA are in the ethylester form.
 6. The method of claim 4, wherein 18-HEPE, 17-HDHA, 14-HDHA,10-HDHA, and 4-HDHA are in the remodeled tri-glyceride form.
 7. Themethod of claim 1, wherein the composition further comprises a compoundselected from the group consisting of: 5S-HEPE(5S-hydroxy-eicosa-6E,8Z,11Z,14Z,17Z-pentaenoic acid), 11S-HEPE(11S-hydroxy-eicosa-5Z,8Z,12E,14Z,17Z-pentaenoic acid), 12S-HEPE(12S-hydroxy-eicosa-5Z,8Z,10E,14Z,17Z-pentaenoic acid), 12R-HEPE(12R-hydroxy-eicosa-5Z,8Z,10E,14Z,17Z-pentaenoic acid), 15S-HEPE(15S-hydroxy-eicosa-5Z,8Z,11Z,13E,17Z-pentaenoic acid), 7S-HDHA(7S-hydroxy-docosa-4Z,8E,10Z,13Z,6Z,19Z-hexaenoic acid), 11S-HDHA(11S-hydroxy-docosa-4Z,7Z,9E,13Z,16Z,19Z-hexaenoic acid), 20S-HDHA(20S-hydroxy-docosa-4Z,7Z,10Z,13Z,16Z,19Z-hexaenoic acid), 17S-HDPAn-6(17S-hydroxy-docosa-4Z,7Z,10Z,13Z,15E-pentaenoic acid), 14S-HDPAn-6(14S-hydroxy-docosa-4Z,7Z,10Z,12E,16Z-pentaenoic acid), 10S-HDPAn-6(10S-hydroxy-docosa-4Z,7Z,11E,13Z,16Z-pentaenoic acid), 17S-HDPAn-3(17S-hydroxy-docosa-7Z,10Z,13Z,15E,19Z-pentaenoic acid), 14S-HDPAn-3(14S-hydroxy-docosa-7Z,10Z,12E,16Z,19Z-pentaenoic acid), 10S-HDPAn-6(10S-hydroxy-docosa-4Z, 7Z,11E,13Z,16Z-pentaenoic acid), 15S-HETE(15S-hydroxy-eicosa-5Z,8Z,11Z,13E-tetraenoic acid).
 8. The method ofclaim 1, wherein said administration lowers macroscopic and physicalsigns of inflammation.
 9. The method of claim 1, wherein saidadministration treats pain relating to an inflammatory condition. 10.The method of claim 1, wherein said administration stimulates resolutionof inflammation.
 11. A method of stimulating resolution of inflammationin a subject in need thereof, the method comprising administering to thesubject an effective amount of a composition comprising fatty acidsincluding 18-hydroxy-eicosapentaenoic acid (18-HEPE),17-hydroxy-docosahexaenoic acid (17-HDHA), 14-hydroxy-docosahexaenoicacid (14-HDHA), 10-hydroxy-docosahexaenoic acid (10-HDHA),4-hydroxy-docosahexaenoic acid (4-HDHA), eicosapentaenoic acid (EPA) anddocosahexaenoic acid (DHA), wherein the fatty acids are in ethyl esterform and the 17-HDHA is present in an amount of at least 100 mg/L. 12.The method of claim 11, wherein the composition further comprises acompound selected from the group consisting of: 5S-HEPE(5S-hydroxy-eicosa-6E,8Z,11Z,14Z,17Z-pentaenoic acid), 11S-HEPE(11S-hydroxy-eicosa-5Z,8Z,12E,14Z,17Z-pentaenoic acid), 12S-HEPE(12S-hydroxy-eicosa-5Z,8Z,10E,14Z,17Z-pentaenoic acid), 12R-HEPE(12R-hydroxy-eicosa-5Z,8Z,10E,14Z,17Z-pentaenoic acid), 15S-HEPE(15S-hydroxy-eicosa-5Z,8Z,11Z,13E,17Z-pentaenoic acid), 7S-HDHA(7S-hydroxy-docosa-4Z,8E,10Z,13Z,6Z,19Z-hexaenoic acid), 11S-HDHA(11S-hydroxy-docosa-4Z,7Z,9E,13Z,16Z,19Z-hexaenoic acid), 20S-HDHA(20S-hydroxy-docosa-4Z,7Z,10Z,13Z,16Z,19Z-hexaenoic acid), 17S-HDPAn-6(17S-hydroxy-docosa-4Z,7Z,10Z,13Z,15E-pentaenoic acid), 14S-HDPAn-6(14S-hydroxy-docosa-4Z,7Z,10Z,12E,16Z-pentaenoic acid), 10S-HDPAn-6(10S-hydroxy-docosa-4Z,7Z,11E,13Z,16Z-pentaenoic acid), 17S-HDPAn-3(17S-hydroxy-docosa-7Z,10Z,13Z,15E,19Z-pentaenoic acid), 14S-HDPAn-3(14S-hydroxy-docosa-7Z,10Z,12E,16Z,19Z-pentaenoic acid), 10S-HDPAn-6(10S-hydroxy-docosa-4Z, 7Z,11E,13Z,16Z-pentaenoic acid), 15S-HETE(15S-hydroxy-eicosa-5Z,8Z,11Z,13E-tetraenoic acid).
 13. The method ofclaim 11, wherein said administration lowers macroscopic and physicalsigns of inflammation.
 14. The method of claim 11, wherein saidadministration treats pain relating to an inflammatory condition.